For over 200 million years, milk has co-evolved with mammalian infants to be nourishing and immunoprotective.
Scientists are now realizing that milk is not simply a mixture of basic nutritional building blocks, but rather a dynamic,
active and personal source of nourishment to the human infant. Fragments of milk proteins released during infant
digestion have an array of biological functions within the gut and systemically including opioid-like activity, immune
system modulation and antimicrobial action. These peptides are typically inactive within the sequence of the parent
protein and only become active when released by proteolysis.
For the majority of mammalian evolution, milk evolved with term-delivered infants as the digestive partner because
historically most premature infants (infants born at <37 weeks of gestation in humans) did not survive. Only through modern medical interventions do the majority of premature infants now survive to adulthood. Thus, premature infants have had no influence on the evolution of human milk. In essence, bioactive peptide fragments evolved to be released in the digestive milieu of the term infant. Premature infants have a reduced capacity for protein digestion in comparison with term infants. A reduced proteolytic capacity means that biologically active milk protein fragments released by the specific digestive milieu of the term infant may not be released in the preterm infant. Therefore, the premature infant may not obtain the benefit of the peptide fragments’ biological functions. The absence of peptide-induced biological functions in the gut could be partially responsible for the increased risks of morbidity found in preterm infants compared to term infants. Therefore, in the project, breast milk from mother’s of term and premature infants and gastric contents of infants two hours post-feeding are analyzed for presence of protein fragments by nano-LC Chip Quadrupole Time-of-Flight Tandem Mass Spectrometry (Chip-Q-TOF). Tandem-MS spectra are analyzed with the Global Proteome Machine against a human milk protein library compiled from literature. For each tandem-MS analysis, the list of identified peptides is used as an exclusion list in a subsequent round of tandem analysis to prevent re-fragmentation of the same peptides and to allow further peptide discovery. Tandem analysis is repeated with this iterative exclusion lists strategy until few new peptides are identified. We first investigated peptides in term human milk, as milk contains a variety of active proteases. After the peptide fragments that exist in vivo are identified, we use computational methods to compare the sequences to functional domain databases to determine hypothetical peptide function. From term human breast milk, we have identified 639 unique milk peptides, most of which were derived from β-casein. Other peptides were derived from osteopontin, κ-casein, αs1casein, immunoglobulins κ and γ, butyrophilin, polymeric immunoglobulin receptor, macrophage mannose receptor-1 and mucins as well as 65 other milk proteins. Notably, no peptide fragments from the major milk proteins lactoferrin, α-lactalbumin and secretory immunoglobulin A were identified. Phosphorylation—a prevalent modification among human milk proteins—was present in 22% of the peptides. Some milk protein phosphorylation sites were identified for the first time. Sixty peptides had significant sequence overlap with known bioactive peptides with antimicrobial or immunomodulatory functions.